The invention relates to the radiopharmaceutical labeling of diseased or malfunctioning candidate cells for subsequent treatment with a medicinal compound, the identification in situ of the candidate cells using a probe sensitive to the presence of the radiopharmaceutical label and then treatment of the identified cell. Addressed are improved instruments with enhanced operability, controllability, diagnostic capability and treatment capability. For example, the described devices can be used in an MRI environment, can provide a visual image as well as a radiation image, allow 3D imaging, and allow delivery of treatment compounds to the cell while the probe is at the site of the labeled cells. In one embodiment, the invention relates to in situ gene therapy using a beta or gamma probe to locate labeled cells, also referred to as candidate cells, and the delivery of corrective or therapeutic genes to the candidate cells identified by the probe while the probe is positioned adjacent to the labeled and located cells.
Most of the basic elements of biological materials have radiation emitting isotopes (e.g., C-11, N-13, O-15, F-18, I-124). For example, these compounds can be labeled with isotopes which emit positron, beta or gamma rays. More than 500 biochemicals have been labeled with these isotopes (e.g., amino acids, fatty acids, sugars antibodies, drugs, neuroreceptor ligands, nucleoside analogues, etc).
Recently, several chemical compounds have been labeled with various positron emitting tracer isotopes for the imaging of gene expression. For example I-124 labeled FIAU a 2xe2x80x2-fluoro-substituted nucleotide analogue, and PET studies performed on rats (Tjuvajev et al. Cancer Res 55, 6126-6132 (1995); Tjuvajev et al. Cancer Res 56, 4087-4095 (1996); Tjuvajev et al. Cancer Res. (1999) in Press) [8-F18]-fluoroganciclovir has been used for PET studies of gene transduction in mice (Gambhir et al. J. Nucl. Med. (in Press) (1998); Haberkorn U et al. J Nucl. Med. 38: 1048-1054 (1997)). The goal of these procedures was to introduce radiolabeled tracers after gene therapy to determine if the gene therapy was successful. The presently described invention is fundamentally different because the below described invention entails radiolabeling of cells suitable for gene therapy and providing a gene therapy composition directly to labeled cells while targeted by the probe.
In attempts to locate cancerous cells numerous labeling techniques have been developed to identify the site of those cancerous cells. It was recognized many years ago that fibrin, while not a tumor specific antigen, was known to be more prevalent in the vicinity of tumors due to the inflammatory process accompanying the cell proliferation. Therefore radiolabeled immunoglobulin was used for tumor localization (Day, E. O.; Planisek, J. A.; Pressman D.; xe2x80x9cLocalization of Radioiodinated Rat Fibrinogen in Transplanted Rat Tumorsxe2x80x9d, J. Natl. Cancer Inst. 23: 799-812, 1959. Sparr, J. L.; Bale, W. F.; Marrock, D. D.; Dewey, W. O.; McCardle, R. J.; Harper, P. V.; xe2x80x9cLabeled Antibodies to Human Fibrinogen. Diagnostic Studies and Therapeutic Trailsxe2x80x9d, Cancer, 20: 865-870, 1967.) In all these works the goal was to use radiolabled tracers after gene therapy to determine if the gene therapy was successful. The new method described in this patent application is fundamentally different because radio labeled tracers are used to locate the cells that are suitable candidates for gene therapy before the therapy is applied.
More specific labeling was accomplished by Goldbenberg, et al. by the use of I131-labelled heterologous (goat) antibodies to human carcinoembryonic antigen (CEA). (Goldenberg, D. M.: xe2x80x9cOncofetal and other Tumor-associated Antigens of the Human Digestive Systemxe2x80x9d, Curr. Top. Pathol. 63: 289-342, 1976. Goldenberg, D. M.; Deland, F.; Kim, E. E.: xe2x80x9cHuman Chorionic Gonadotrophin Radioantibodies in the Radio immunodetection of Cancer and the Disclosure of Occult Metastasesxe2x80x9d Proc. Nat""l. Acad. Sci. 78: 7754-7758, 1981.; Goldenberg, D. M.; Deland, F.; Kim, E. E., et al.: xe2x80x9cUse of Radio labeled Antibodies to Carcinoembryonic Antigen for the Detection and Localization of Diverse Cancers by External Photoscanningxe2x80x9d, N. Engl. J. Med. 298: 1384-1388, 1978.; Goldenberg, D. M.; Preston, D. F.; Primus, F. J.; Hansen, H. J.: xe2x80x9cPhotoscan Localization of GW-39 Tumors in Hamsters Using Radiolabeled Anticarcinoembryonic Antigen Immunoglobulinxe2x80x9d J. Cancer Res. 34: 1-9, 1974.; Goldenberg, D. M.; Sharkey, R. M.; Primus, F. J.: xe2x80x9cCarcinoembryonic Antigen in Histopathology: Immunoperoxidase Staining of Conventional Tissue Sectionsxe2x80x9d, J. Natl. Cancer Inst. 57: 11-22, 1976.) CEA is a tumor-associated antigen of gastrointestinal cancer, particularly colon and pancreatic cancer, first described by Gold. (Gold, P., Freedman, S. O.: xe2x80x9cDemonstration of Tumor Specific Antigen in Human Colonic Carcinomata by Immunologic Tolerance and Absorption Techniquesxe2x80x9d, J. Exp. Med. 121: 439-462, 1965.) Other labeled antibodies usable for tagging tumor cells include monoclonal antibody 17-1A and its F(abxe2x80x2)2 fragment (Wistar Institute, Philadelphia, Pa.), monoclonal antibody 19-9 and its F(abxe2x80x2)2 fragment (Centocor, Inc., Philadelphia, Pa.), monoclonal antibody B72.3 (Dr. Jeffrey Schlom, National Cancer Institute) and CC49 and CC83, both second generation B72.3 antibodies. These are identified as examples of suitable materials and are not meant to limit the scope of compounds usable to label cells. Many other compounds, such as single chained antibodies (SCAs) disclosed in U.S. Pat. No. 4,946,778, capable of labeling specific cells, are identified in the literature and are constantly being discovered and/or developed. Labeling nucleotides detectable by a gamma probe include technetium Tc99,, iodine I123,, I125, and I131, indium In111, selenium Se75, and cobalt Co57. These and other radioisotopes can be detected by beta or gamma probes.
Martin et al., U.S. Pat. No. 4,782,840, incorporated herein by reference, describes a procedure which requires the administration of I125 labeled antibody or antibody fragments to a patient to label cancerous tissue. Some time after administration (2 to 21 days) the suspected site is accessed surgically and, using a hand-held gamma probe, the labeled tissue is located and surgically removed.
Applicant is a co-inventor on U.S. Pat. Nos. 5,008,546, 5,325,855 and 5,338,937, which describe and claim variations to prior know intraoperative radiation probes. Others describe the use of gamma probes as a biopsy probe for locating, localizing or mapping tagged tissue located throughout the body and particularly near the liver, kidney, or blood vessels or to localize lymph nodes (U.S. Pat. Nos. 4,959,547, 5,170,055 and 5,036,201 to Carroll et al; U.S. Pat. No. 5,383,456 to Arnold et al.). Leone et al, U.S. Pat. No. 5,811,814 describes a catheter, including fiber optics and a scintillation crystal, suitable for locating concentrations of alpha, beta, gamma or X-ray labeled compounds introduced into the arteries and veins.
The use of radiation detection probes placed through scopes to locate radionuclide labeled tissue has been described in the literature for many years. Both Barber et al and Woolfenden et al. described the insertion of a gamma ray detection probe through an open channel in a broncoscope. (Barber, H. B., Woolfenden, J. M., Donahue, D. J., Nevin, W. S., xe2x80x9cSmall Radiation Detectors for Bronchoscopic Tumor Localizationxe2x80x9d, IEEE Transactions on Nuclear Science, NS-27, No. 1 Febuary 1980; Woolfenden, J. M., Nevin, W. S., Barber, H. B., Donahue, D. J., xe2x80x9cLung Cancer Detection Using a Miniature Sodium Iodide Detector and Cobalt-57 Bleomycinxe2x80x9d, Chest, 85, 1, January 1984). Goldenberg, U.S. Pat. No. 4, 932,412, issued Jun. 12, 1990 claimed the same technique, namely the use of a radiation detection probe placed through an endoscope to locate radionuclide labeled tissue.
U.S. Pat. No. 5,846,513 to Carroll et al. describes a probe for percutaneous insertion into a body through a delivery sheath followed by the removal of the probe and insertion through the same sheath of an instrument, such as a resectoscope, to remove the identified tissue. Alternatively, the ""513 patent discloses that, following removal of the probe, other tumor destroying techniques can be practiced by delivering a treatment media or device through the sheath, such as cancer cell necrotizing agents, high intensity ultrasound, microwave energy, laser energy, heat electrocoagulation, or the introduction of tumor destructive chemical agents such as free radical promoters, copper or iron ions, oxidants, iodine, tissue digestive enzymes, alcohol or radioactive seeds. However, Carroll et al did not suggest the delivery of compositions for gene therapy which, as discussed below, function in a fundamentally different manner from chemical, mechanical or electrical tumor destruction techniques.
U.S. Pat. No. 5,014,708 discloses a device insertable within the body which includes in combination, a radiation sensing probe with an ultrasonic tip and aspiration function to remove tissue released by the vibrating ultrasound tip. U.S. Pat. No. 4,995,396 sets forth an endoscope which includes, in combination, a radiation detecting probe with means to deliver tumor affinable chemicals which can then be activated by laser light transmitted through fiber optics also enclosed within the endoscope. Neither patent suggests gene therapy or the other new functions described below.
Another technique attempted to treat cancer is adoptive immunotherapy. Using lymphokines such as Interlukin-2 (IL-2) and lymphokine-activated killer cells (LAK) derived from patient peripheral blood, patients with melanoma and renal cell cancer have shown a significant positive response. A related approach is the in-vitro placement of cytokine genes into tumor specific lymphocytes. After a few days the cytokine gene supplemented lymphocytes are delivered locally to a tumor.
Rosenberg, et al. demonstrated that a small but significant percentage of patients with melanoma and renal cell cancer could achieve a long-lasting response. (Rosenberg, et al., xe2x80x9cAdoptive Cellular Therapy: Clinical Applicationsxe2x80x9d, Biologic Therapy of Cancer, De Vita, et al. (Eds.), J. B. Lippincott Company, Philadelphia, Pa., 1991.) A second approach to adoptive immunotherapy is to expand lymphocytes from tumors in culture. (Rosenberg, et al., xe2x80x9cAdoptive Cellular Therapy: Clinical Applicationsxe2x80x9d, Biologic Therapy of Cancer, De Vita, et al. (Eds.), J. B. Lippincott Company, Philadelphia, Pa., (1991); Topalian, et al. xe2x80x9cTumor Infiltrating Lymphocytes: Evidence of Specific Immune Reactions Against Growing Cancers in Mice and Humanxe2x80x9d, Important Advances in Oncology 1990, De Vita, et al. (Eds.), J.B. Lippincott Company, Philadelphia, Pa., p. 19 (1990), and Rosenberg, et al., xe2x80x9cUse of Tumor-Infiltrating Lymphocytes and Interleukin-2 in the Immunotherapy of Patients with Metastatic Melanomaxe2x80x9d, N. Engl. J. Med., 25: 1671, 1988.) Using these tumor-infiltrating lymphocytes (TIL), several research groups have documented superior tumor cytolytic activity and better delivery of these TIL cells to tumors than LAK cells. (Rosenberg, et al., N. Engl. J. Med., id.; Diliman, et al., xe2x80x9cContinuous Interleukin-2 and Tumor-Infiltrating Lymphocytes as Treatment of Advanced Melanomaxe2x80x9d, Cancer, 68: 1, 1991; Kradin, et al., xe2x80x9cTumor-Infiltrating Lymphocytes in Interleukin-2 in Treatment of Advanced Cancerxe2x80x9d, Lancet, 33: 577, 1989; and Bukowski, et al., xe2x80x9cClinical Results and Characterization of Tumor-Infiltrating Lymphocytes with or without Recombinant Interleukin-2 in Human Metastatic Renal Cell Carcinomaxe2x80x9d, Cancer Res. 51: 4199, 1991.) In general, TIL Cells appear to be therapeutically effective for patients with melanoma. Tumor-infiltrating lymphocytes have been generated from many solid tumors, including colon and breast cancer; however, these cells do not appear to mediate tumor-specific cytolytic activity in vitro and it is not known if these cells will be effective in adoptive immunotherapy models. (Rosenberg, xe2x80x9cGene Therapy of Cancerxe2x80x9d, Important Advances in Oncology, 1992, De Vita, et al. (EDS.), J.B. Lippincott Co., New York, N.Y., pp 17-18, 1992.)
Another approach to tumor therapy with tumor-specific lymphocytes is the placement of cytokine genes in cells which can deliver cytokines locally to the tumor. (Kasid, et al., xe2x80x9cHuman Gene Transfer: Characterization of Human Tumor Infiltrating Lymphocytes as Vehicles for Retroviral-Mediated Gene Transfer in Manxe2x80x9d, Proc. Natl. Acad. Sci. USA, 87: 473-477, 1990; and Rosenberg, et al., xe2x80x9cGene Transfer into Humans: Immunotherapy of Patients with Advanced Melanoma Using Tumor Infiltrating Lymphocytes Modified by Retroviral Gene Transductionxe2x80x9d, New Engl. J. Med., 323: 570-578, 1990.) It has been shown in several model systems that tumor cells transfected with various cytokine genes including IL-2, gamma interferon, and tumor necrosis factor (TNF), are more immunogenic and less tumorigenic than parent cells that do not produce cytokines. (Gansbacher, et al., xe2x80x9cRetroviral Vector-Mediated Gamma Interferon Gene Transfer into Tumor Cells Generates Potent and Long Lasting Antitumor Immunityxe2x80x9d, Cancer Res. 50: 7820-7825, 1990; Gansbacher, et al., xe2x80x9cInterleukin2 Gene Transfer into Tumor Cells Abrogates Tumorigenecity and Induces Protective Immunityxe2x80x9d, J. Exp. Med., 172: 1217-1224, 1990; and Blankenstein, et al., xe2x80x9cTumor Suppression after Tumor Cell-Targeted Tumor Necrosis Factor-Alpha Gene Transferxe2x80x9d, J.,Exp. Med. 173: 1047-1052, 1991.)
It appears that local production of cytokines near tumor cells can inhibit tumor growth and stimulate an immune response. It would therefore appear useful to find lymphocytes that recognize tumors and are capable of secreting various cytokines in response to tumors and to deliver these lymphocytes to labeled tumor cells for adoptive immunotherapy. It has been shown that certain TIL cells that secrete gamma-interferon and TNF-alpha will cause tumor regression in vivo, even though they do not display direct tumor cytotoxicity in vitro. (Barth, et al., xe2x80x9cInterferon-Gamma and Tumor Necrosis Factor Have a Role in Tumor Regression Mediated by Murine. CD8+ Tumor-Infiltrating Lymphocytesxe2x80x9d, J. Exp. Med., 173: 647, 1991.)
An alternative source of tumor lymphocytes is lymph nodes. Martin et al., U.S. Pat. No. 5,814,295, describes a method of locating, within cancer patients, lymph nodes enriched in tumor reactive lymphocytes so these cells can be harvested, cultured and delivered to the donor patient. The method comprises administering to the patient a radiolabeled locator (such as an antibody) which, in addition to concentration in cancer tissue, also concentrates in the lymph nodes, which are rich in tumor reactive lymphocytes. A gamma probe is then used to locate lymph nodes with increased radiation levels and those nodes are surgically excised. Nodes that appear normal (i.e. free of gross metastatic disease) but which took up the radiolabeled antibody are separated and cultured to proliferate tumor reactive cells with tumor-specific T lymphocytes therein. The cultured tumor reactive cells can then undergo gene therapy in-vitro as described above and, following a several day incubation period, transfused into the patient in accordance with adoptive immunotherapy regimes. This is fundamentally different from the invention described herein where the tumor reactive cells are delivered in vivo to the tumor and allowed to attack the tumor cell within the body of the cancer patient.
Gene therapy involves the insertion of genes or parts of DNA into cells or the cell membrane such that they become part of the genetic structure of the cell. Typically, a DNA vector capable of expressing a suitable gene product in the cells of the target organism is transferred into the cells of the organism, through one of a variety of processes so that it interacts with the genetic material of the cell. Prior art mechanisms for the insertion of genetic material into living tissues include direct microinjection, electroporation, (a technique in which individual cells are subjected to an electric shock to cause those cells to uptake DNA from a surrounding fluid), liposome-mediated transformation, (DNA or other genetic material is encapsulated in bilipid vesicles which have an affinity to the cell walls of target organisms), and the use of specific types of biological vectors or carriers which have the ability to transfect genetic material carried within them into specific target organisms. For example, Nemunaitis et al reports on the beneficial effects of the direct injection into tumors in the lung of Adp 53 in combination with cisplatin (Nemunaitis, J, et al xe2x80x9cAdenovirus-Mediated p53 Gene Transfer in sequence with Cisplatin to Tumors of Patients with Non-Small-Cell Lung Tumor,xe2x80x9d J.Clin. Oncology, 18, No 3, (Febuary, 2000) p609-622.
One general technique applicable to a large range of hosts is referred to as particle mediated genetic transformation. In this technique, the genetic material, (RNA or DNA) is coated onto small carrier particles. The particles are then accelerated toward target cells where the particles impact the cells and penetrate the cell walls, carrying the genetic material into the cells. At least a proportion of the cells into which the genetic material is delivered express the inserted genetic material and another smaller proportion of the cells may integrate the delivered genetic material into the cells native genetic material.
One method of accelerating coated carrier particles utilizes a larger carrier object, sometimes referred to as a macroprojectile. The carrier particles are positioned inside the macroprojectile. The macroprojectile is then accelerated at a high speed toward a stopping plate. One means of accelerating the microprojectile is to use a gunpowder driven device in which the hot gases generated by a gunpowder discharge form a hot gas shock wave, which accelerates the macroprojectile. When the macroprojectile strikes a stopping plate with a hole therein, the microprojectiles continue their travel through the hole and eventually strike the target cells. This and other acceleration techniques have been described in U.S. Pat. No. 4,945,050 issued to Sanford et al. and entitled xe2x80x9cMethod For Transporting Substances Into Living Cells And Tissues And Apparatus Thereforexe2x80x9d incorporated by reference herein.
A second technique developed for the acceleration of carrier particles is based on a shock wave created by a high voltage electric spark discharge. This technique involves an apparatus having a pair of spaced electrodes placed in a spark discharge chamber. The high voltage discharge is then passed between the electrodes to vaporize a droplet of water placed between the electrodes. The spark discharge vaporizes the water droplet creating a pressure wave, which accelerates a carrier material previously placed in the discharge chamber. The carrier transports the particles which are coated with the genetic materials to be delivered. The carrier is accelerated toward a retainer, where it is stopped, the particles are separated from the carrier, and the particles carried thereby pass on into the biological tissues.
This second technique has been incorporated into a hand-held device that can be use for accelerating particles carrying biological materials into large whole organisms. The hand held device is described in U.S. Pat. No. 5,149,655 to an xe2x80x9cApparatus For Genetic Transformationxe2x80x9d. issued to McCabe et al.
A variation on the second technique for acceleration of carrier particles is based on an expanding gas shock wave, and a planar surface having carrier particles positioned on the target side of the planar surface. The shock wave that actually impacts the target area is substantially reduced when this technique is utilized. In addition, the apparatus used with this technique does not subject target cells to radiant heat or appreciable acoustic energy. Hence cell differentiation and successful cell transformation is maximized. This technique is described in U.S. Pat. No. 5,204,253, entitled xe2x80x9cMethod and Apparatus For Introducing Biological Substances Into Living Cells.xe2x80x9d which issued to Sanford et al.
In this third technique, the delivery instrument incorporates a high pressure gas delivery system, a mechanism to generate an instantaneous gas shock out of the high pressure system, an enclosure into which the gas shock is released, contained and vented and a throat region which translates the gas shock into a particle acceleration force. The expanding gas shock is directed at, and impacts on, a back surface of the planar insertion mechanism (the carrier particles being on the front surface of the insertion mechanism). The particles are then disbursed from the front surface over a wide region of the target cells.
All of the techniques discussed can generate only a single potentially traumatic, essentially instantaneous burst of carrier particles and thus are single shot insertion devices. In order to utilize the single shot apparatus a second time, a new carrier with genetic material thereon must be inserted into the device.
U.S. Pat. No. 5,525,510 is directed to an apparatus for injecting a continuous stream of carrier particles carrying genetic material into living cells. It includes a body member having an acceleration channel along a central axis, with the channel having an outlet at an exit end. The body also includes a source chamber connected to a compressed gas source and to the channel. Particles are placed on a carrier mounted in the body member in a position exposed to the channel so that a gas stream flowing in the channel can pick up carrier particles off of the particle carrier. A gas stream diverter is placed on the body adjacent the outlet of the channel diverts the gas stream away from the direction of flight of the carrier particles as they exit the body. In this manner a continuous stream of particles carrying genetic material can be directed to the target cells.
It has been discovered that cancer cells may have a defective gene structure. It is believed that correction of this gene structure, or modification of that structure can convert the cancerous cell to a normal cell or, as a minimum, modify or retard the cancerous characteristics of those cells and prevent the proliferation of the cancer cells. Alternatively, cancer cells can be transfected by DNA that makes them undergo the apoptosis process. Gene therapy is not limited to cancer cells as it is believed that a gene defect can be identified in other abnormal cells. Also, genes can be delivered to the cell to cause the cell to then generate therapeutic substances. The inventive devices described herein can localize the schemic tissues and allow the effective and accurate insertion of therapeutic genes. There are several radiolableled compounds that can be accumulated in schemic tissue. For example, cells undergoing the apoptotic process can be targeted. Additionally, genes can be inserted in myocardial cells causing them to generate angiogenic factor, which promote the generation of blood vessels.
It has also been recognized that diseased cells or tissue can be radiolabeled using various cell specific, tagged compounds. It is also known that these labeled cells or labeled tissue can be located using a probe containing a radiation sensitive detector mounted to receive emissions from the radiolabeled tissue. However, no one has provided a readily usable means for delivering, in vivo, corrective genes directly to the labeled (candidate) tissue in the patient""s body while that tissue is under surveillance by the probe.
The invention comprises a probe for the in vivo location of radiolabeled cells or tissue using a radiation sensitive crystal or other detector devices, such as semiconductor detector materials, mounted in the tip of a tubular device insertable into a mammalian body to the region of the suspected radiolabeled cells. As used herein, each reference to xe2x80x9cradiolabeled cellsxe2x80x9d alternatively is a reference to radiolabeled tissue or tissue containing radiolabeled cells. The device includes channels therein, or associated therewith, for placement of compositions for modifying, in a desired manner, the radiolabeled cells directly on or in the labeled cells, or in the immediate vicinity of the labeled cells, while the probe is targeting the labeled cells. The invention also includes a method for gene therapy comprising marking abnormal cells or abnormal tissues with a radioactive tag, targeting those tagged cells using a probe directionally sensitive to the proximity of radionuclides and, through a channel placed at the site of the target cells along with the probe, delivering genetic material prepared for gene therapy treatment purposes.
The invention also contemplates the use of a collimating material unaffected by, or not inferring with, a magnetic field, such as a gold collimator, so the probe can be used intraoperatively within the field of an MRI diagnostic instrument.
Further, devices within the scope of the invention can include fiber optic channels, which will allow illumination and/or optical visualization of the targeted tissue at the same time a radiation image is being generated. More then one fiber optic channel will allow a 3D image to be generated.
Still further, devices within the scope of the invention can also include position-locating means attached to the distal end of the detector. This allows the location of the detector tip within the patient""s body to be continuously monitored and recorded and, simultaneously, a visual image of targeted tissue recorded with a radiation count from the labeled tissue superimposed digitally thereon. This will provide a contemporaneous viewable image of the labeled tumor, an audible indication of radiation intensity across the targeted site, and a digital visual record of the procedure.
It should be recognized that each of these concepts, namely
a) the delivery of gene therapy
b) use of the probe in an MRI field
c) mapping of the operative site, and
d) the generation of an optical and radiation image may be utilized independently or two or more of these concepts may be combined in a single device.